Search

Objective In order to analyze morphologic processes occurring during development of mammalian CNS, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in neurons. In this study, we have determined and compared pattern of Thy1 - YFP expression during embryonic development as well as during differentiation of neural stem cells culture. Material and methods Pregnant females, at different stages of gestation were used for embryo isolation. The embryos were fixed and cryosectioned on 20µm thick slices. Neural stem cells were isolated from the forebrain of 14.5 old murine embryos and cultured as neurospheres. After dissociation, cells were plated on cover slips. The cells were fixed 1, 3, 5 and 7 and prepared to the immunocytochemistry. Results First signs of expression were observed at embryonic day 12.5 in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in neurons after E12.5 became much more pronounced. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. Furthermore, expression of YFP was determined during in vitro NSC differentiation. YFP expression was observed in neural progenitors as well as in mature neurons. YFP positive cells stained positive for Map2 (mature neuronal marker), while astrocytes which were GFAP positive were not expressing YFP. Conclusion Our results showed that, in addition to mature neurons, neuronal progenitor cells express this construct both in in vivo and in vitro conditions. This suggests that in addition to analyses of neuronal differentiation in B6.Cg- Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed.

The mouse transcription factor Noto is expressed in the notochord and involved in its development. Noto mouse mutants, Noto(tc/tc)(truncate) and Noto(GFP/GFP) (Noto null mutant), exhibit a segmental lack of the notochord in the caudal part of the embryo and subsequent tail truncation in adult animals. In order to address the relationship between the tail bud (the undifferentiated mesenchymal cells in the tip of the embryo tail) and the caudal notochord, Noto(GFP/GFP), a loss- of-function mutant, was analyzed. Taking advantage of Noto(GFP/+) heterozygotes, we could track Noto-GFP-expressing cells from the tail bud over the tail cord to the caudal notochord, and confirm a morphological continuum from the tail bud mesenchyme to the caudal notochord. Loss of Noto affected tail bud morphogenesis: Noto-GFP-expressing cells were scattered in the tail bud mesenchyme. They segregated in the notochord- like structure within the medullary cord instead at the tail cord, which subsequently disappeared. In the tail cord, instead of the notochord, additional lumen of the tail gut was formed. These findings suggest that Noto is involved in both rearrangement and morphogenesis of the tail bud during notochord formation.

Analysis of expression of Stam2 gene using a gene trap derived transgenic mouse model

Expression analysis of Stam2 gene in the brain using a gene trap derived transgenic mouse model

The morphological analysis represents the basic method used to determine the phenotype changes of the genetically modified mouse. In this way the observed phenotype changes are correlated to the genetic modification and enable the insight in the function of the investigated gene. The challenge of the morphological analysis increases with the increased complexity of the investigated tissues and organs. Therefore the most complex organ to be studied is the brain. In order to compare the brain neuroanatomy of the mutant and wild type mice, the brain could be studied at the gross-morphology level as a whole, at the light microscope level on histological sections, or at the ultrastructural level by electron microscopy (1). In order to compare the brain morphology at the ultrastructural level of the mutant and the wild type mouse, we used the mouse mutant Stam2Gt1Gaj, where due to the gene trap modification the function of Stam2 was hindered. Due to the brain size and the nature of electron microscopy analyisis only two brain regions were selected to be viewed by electron microscope, CA2 region of the hippocampus and the adjacent cerebral cortex. In order to compare the equivalent regions of the brain in the mutated and wild type mice the regions of interest were determined on vibratome sections, and the selected pieces of tissues were processed for electron microscopy. Adult, 3 months old mice were anesthetized with 1% avertin and perfusion fixation was performed. 0.1 M phosphate buffer was injected within the left heart ventricle in order to rinse the blood from the circulatory system. Afterwards, the animals were perfused with 12.5% glutaraldehyde, which resulted with initial fixation of the brain. Skull was opened with sharp scissors and the brain was removed and immersed in 6% glutaraldehyde for 1 hour. The brain was afterwards cut in 200 µ m thick sagital sections using vibratome. After determination of the brain regions of interest, the tissue was washed in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide for 2 hours. Subsequent to contrasting in uranyl acetate over night, the tissue was dehydrated in ethanol and embedded in Durcopan (Fluka). 1 µ m thick semithin sections were cut using the ultramicrotome and examined by light microscopy (figure 1). The selected and comparable regions of the semithin sections were trimmed and 70nm thick ultrathin sections were made, contrasted with lead citrate and uranyl acetate and examined by electron microscopy on transmission electron microscope Zeiss902A (2) (figure 2). The described procedure enabled the comparison of the brain ultrastructure of mice with different genetic background. The comparison was complemented with the photographing of the serial frames of the selected regions in order to increase the size of area for analysis and acquire the necessary data to be used in image analysis applications. Nevertheless, the variability of the tissue examined should be add to the variability among the mice belonging to the same genotype. The presented example highlights the difficulties of morphology analyisis of the genetically modified mice.

Endosomes are involved in the CNS in a variety of actions including formation of synaptic vesicles, axonal transport and receptor sorting. To elucidate function of Stam2 in the CNS, gene trap approach was applied. The expression pattern analysis showed that Stam2 was expressed in developing neural tube and heart. In addition, alternatively spliced variant of Stam2 was found.

STAM2 (Signal transducing adaptor molecule 2) is a signaling adaptor presumed to act in the intracellular cargo sorting for degradation at the level of the late endosome (1). Although its function was already assessed at the cellular level in permanent cell cultures, its function in mammalian organism could be revealed only through genetic modification at the level of the whole animal. In order to characterize Stam2 function in mouse, the gene trap method was applied. It comprises the genetic modification of embryonic stem cells with a non-homologous vector containing a splice acceptor and fused lacZ and neoR genes without their own promoters. In case the vector was inserted in an endogenous gene, the splice acceptor would be recognized and lacZ and neoR genes would be driven by the promoter of the endogenous gene. This enabled to screen the genetically modified embryonic stem cells and the corresponding mouse lines for lacZ activity, which mirrored the expression of the endogenous gene. In addition the vector insertion disturbed the proper protein translation 3’ to the gene trap vector. After the selection process, the mouse line carrying the gene trap mutation in Stam2 gene was chosen for further characterization. The molecular analysis defined the exact place of the insertion and its consequences on the mRNA and the protein level. This showed that the gene trap vector was between exon 2 and 3, and the STAM2 protein production in the homozygous mice was highly diminished (2). Stam2 expression pattern was assessed by monitoring lacZ activity in the investigated tissues. lacZ gives rise to beta-galactosidase, which changes the color of its substrate X-gal in blue. Stam2 expression started in 8.5-day embryos in the hind gut. In 9.5-day embryos it was present in the heart, while in 10.5-day embryos appeared in the ventral part of the neural tube (Figure 1). The expression in the heart persisted during embryo development and continued in the adults. The expression in the neural tube was continuous to the expression in the brain, in particular from 16.5-day embryos in the brain cortex, and from 18.5-day embryos in the hippocampus. In addition to the brain and the heart, Stam2 was expressed as well in the testis and developing adrenal and pituitary glands. In order to assess Stam2 function the phenotype of the homozygous carriers of gene trap mutation was compared with the wild type mice. The homozygotes were alive, fertile and did not show any major abnormalities. The functional studies of mouse behavior were combined with detailed morphological analysis of embryos and adult organs and tissues. The morphological studies involved the classical histology using the hematoxylin-eosin staining, the semithin sections stained by toluidine blue, immunohistochemistry of the selected markers, and finally the ultrastructural analysis using electron microscope (Figure 2). These studies are expected to reveal the fine differences in phenotypes of the wild type and the mutant mice, which would then reveal the Stam2 function in mouse, in particular in the mouse brain.

Development of the caudal part of the mammalian embryo includes differentiation of mesenchymal cells, named the tail bud, in the posterior end of the tail. The tail bud is continuous with the neural tube, notochord and the tail gut, which led to the conclusion that caudal structures of the embryo emerge from the tail bud (1). As experimental manipulations within mammalian embryos are limited by intrauterine location and small dimensions of the embryo, mouse mutants are invaluable tool for investigation of developmental mechanisms. Development of the caudal part of the truncate and splotch mouse mutants, where tail development is affected, was analyzed. Splotch is a spontaneous Pax3 gene mutant, while truncate is a spontaneous Noto gene mutant. Splotch homozygotes die during embryo development showing neural tube, neural crest and somite malformations (2). Corresponding mutation in human causes Waardenburg syndrome characterized by defects of head and hand development and localized absence of pigmentation. Truncate homozygotes have truncated tail, due to the partially absent notochord (3). Splotch and truncate homozygous embryos and wild type control C57BL/6 embryos aged from 9.0 to 12.5 days were isolated, fixed in a mixture of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide. They were embedded in Durcopan (Fluka) and serial semithin sections, perpendicular to the longitudinal tail axis, were obtained, stained with toluidine blue and examined by light microscopy. Splotch embryos from 9.0 to 12.5 days showed open neural tube in the embryo tail. In the 9.0 to 11.5 days old embryos development of other axial structures was not influenced by abnormal neural tube. All axial structures were normal and continuous with the tail bud. In contrast, splotch 12.5 days old embryos showed open neural tube accompanied with disturbed development of other axial structures and disturbed morphology of the tail bud. It is suggested that a longer presence of open neural tube influenced development of all structures caudal from the defect. Truncate homozygous embryos exhibited partially disturbed development of the notochord, which was not continuous to the tail bud, but to the ventral neuroepithelium. In addition, discontinuous or, even doubled notochord was found. Lack of the notochord in the caudal part of the tail was accompanied with disturbed development of the neural tube and the somites. Neural tube was smaller in diameter, it lacked lumen and the massive cell death was found in the neuroepithelium. In the parts of the tail without the notochord, the somites were connected in the midline of the tail. It is suggested that disturbed development of the tail bud is a major cause of truncate phenotype. This investigation confirmed continuity of the tail bud and the tail axial structures in the mouse. The observed malformations indicate that the normal sequence of tail development requires the coordinated interactions among the tail axial structures, which can be related to pathogenesis of spina bifida in humans.

One approach to assess gene function in vivo is to create a mouse deficient for the gene of interest. In order to identify genes expressed during central nervous system development and in the same time to create a mutant mouse, the gene trap method was applied. Embryonic stem (ES) cells were genetically modified by a nonhomologous DNA vector containing a splice acceptor and fused promoterless genes lacZ and neoR. The vector was integrated randomly within the genome, and the inserted genes were active only if the vector was within a transcribed endogenous gene. As a result ES cells were resistant to neomycine (G418) selection. Moreover, expression of lacZ gene mirrored the expression of endogenous gene mutated by gene trap vector, hence its expression could be monitored in ES cells, but as well in ES-cell-derived mutant mice. The insertion was likely to disturb the endogenous gene and an insight in its function could be obtained by analyze of the phenotype of the mutated mice. Here we report the gene trap modification of two genes expressed in developing and adult brain. Signal transducing adaptor molecule 2 (Stam2) is expressed during development of the brain and the heart, and in the adult cortex and hippocampus. The phenotype analysis showed that the homozygous carriers of gene trap modification did not exhibit any obvious anomalies, they were fertile and several generations of homozygotes were obtained. Nevertheless as double knockout of Stam1 and Stam2 is embryonic lethal, Stams appear indispensable for embryo development. Nucleolar protein 1 (Nol1) is expressed in the nucleolus of proliferating cells, including those of the developing neural tube. Homozygote carriers of gene trap mutation of Nol1 gene die at the blastocyst stage. Although Nol1 expression is assigned to dividing cells, it is surprisingly expressed as well in the neurons of the adult brain, suggesting an additional unknown function in the nucleolus of neurons.

Spina bifida is one of the most common congenital defects in humans, as it affects 0, 1% of newborns. Different types of this malformation, from the benign spina bifida occulta to the disabling myelomeningocela have common embryological base: defect of the neural tube closure during embryo development. Spina bifida affects lumbosacral region, i.e. region of junction between primary and secondary neurulation, which involve different morphogenetic mechanisms. In the same way development of the cranial part of the embryo differs from the caudal part. The cranial part is derived from three germ layers created during gastrulation (primary mechanism of embryo development), and the caudal from a mass of undifferentiated cells in the posterior end of the tail, named tail bud. In the cranial direction cells of the tail bud differentiate into neural tube, notochord and the tail gut, while paraaxial mesenchyme segments in the somites (secondary mechanism of embryo development) (1). As the mammalian embryo is difficult to access within the uterus for experimental manipulation, mouse mutants are invaluable tool for investigation of developmental mechanisms including those that take place in the caudal part of the mouse embryo. The main goal of this work was to morphologically describe development of spina bifida in splotch mouse mutant. Splotch was first described in 1947, as a spontaneous mouse mutation of Pax3 gene affecting embryo development (2, 3). Splotch homozygotes die during embryo development showing disorders of neural tube (spina bifida, exencephaly), abnormalities of neural crest cells (absence of sympathetic and spinal ganglia, Schwann cells, melanocytes and cells which migrate to the heart) and defects in somite structure. Corresponding mutation in humans is Waardenburg syndrome: defects of head development (deafness), hand musculature and localized absence of pigmentation. Splotch homozygous embryos aged from 10.5 to 12.5 days were isolated. 11.5 and 12.5 days old embryos were recognized by visible open neural tube. As 10.5 days old homozygotes were not morphologically recognizable, PCR based genotypization of splotch embryos was developed. It included isolation of DNA by isopropanole - ethanole method, constructing of primers specific for mutated and normal Pax3 sequence and polymerase chain reaction. Embryos of all stages were fixed in a mixture of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide. One group of embryos was embedded in Durcopan (Fluka) and serial semithin sections, perpendicular to the longitudinal tail axis, were obtained, stained with toluidine blue and examined by light microscopy. Second group of embryos after fixation and postfixation were dehydrated in ascending mixtures of ethanol and amyl acetate to 100% amyl acetate, dried with liquid CO2, sputter coated with gold and observed in the scanning electron microscope. 11.5 days old splotch embryos showed open neural tube in the middle part of the embryo tail (fig.1 and 2) which is in some cases combined with exencephaly However, development of other axial structures was not influenced by abnormal neural tube, as all axial structures were normal and continuous with the tail bud. In contrast, splotch 12.5 days old embryos showed open neural tube accompanied with disturbed development of other axial structures and disturbed morphology of the tail bud. To find exact location of the onset of spina bifida, 10.5 days old splotch mouse embryos were isolated. It allowed us to find that spina bifida in splotch mutants originates in the caudal part of the neural tube. Described finding that spina bifida originates in the region of secondary neural tube, i.e tissue derived from the tail bud, may lead to explanation that spina bifida is a defect of transition zone between structures derived by primary and secondary body formation mechanisms. The observed malformations in mice give insight in the morphogenetic mechanisms and consequences of spina bifida in humans.

Stam2 (Signal transducing adaptor molecule 2) was identified as a phosphotyrosine protein involved in cellular response to variety of cytokines and growth factors. Together with Hrs (hepatocyte growth factor regulated tyrosine kinase substrate) and Eps15 (EGFR pathway substrate clone no. 15) it has been implicated in endosome mediated intracellular membrane trafficking. In order to investigate expression and function of Stam2, mouse carrying a gene trap modification of Stam2 gene was created. Gene trap method involved genetical modification of embryonic stem (ES) cells with a nonhomologous DNA vector containing a splice acceptor and fused promotorless lacZ and neoR genes. After a large scale screen of obtained ES cells, corresponding mouse lines were produced, and the line carrying modification of Stam2 gene was selected for further investigation. Stam2 expression during development was detected after E9.5 in the gut, notochord, neural tube and heart. In the nervous system it was located in the floor, roof and basal plates of the developing neural tube, and in the cortex, hippocampus and olfactory bulb of the telencephalon. Toward the end of gestation, Stam2 expression appeared in the testis and ovary, pituitary and adrenal glands, skin and epithelium of oral cavity and tongue. As the expression of Stam2 indicated its possible function in the nervous system, general health and behavior of Stam2gt homozygotes was compared to wild type C57Bl/6 mice. Homozygous mutant males had lower body mass, and both sexes preformed poorly in olfactory test. This indicates possible Stam2 function in regulation of body mass and sense of smell.

A key malformation in the homozygous truncate mouse mutants is a partial lack of the notochord in the embryo tail. In order to analyze if tail bud development was affected by the truncate (tc) mutation, serial semithin sections of tails of the homozygous mutant embryos were compared to the wild type controls. In the wild type embryos morphologically uniform mesenchyme of the tail bud was continuous via the medullary cord to the secondary neural tube, and via the tail cord to the notochord and the gut. In truncate embryos the tail cord was not continuous to the notochord, but to the additional lumen of the tail gut resulting in tail gut duplication. Two tail guts subsequently fused together or the additional one disappeared. If present in the tip of the tail, the notochord in truncate embryos ended near the ventral border of the secondary neural tube. The rest of the tail notochord was fragmented and the posterior ends of the fragments were frequently adjacent or even continuous to the neural tube. We suggest that the improper regionalization of the tail bud, where notochord is associated with the neural tube rather than with the tail gut, is related to the subsequent segmental lack of the notochord in truncate mutants.

no abstract

The morphogenesis of the caudal part of the embryo includes formation of a mass of undifferentiated cells in the posterior end of the tail, named the tail bud. In the cranial direction the tail bud is continuous to the neural tube, notochord and the tail gut, while paraaxial mesenchyme segments in the somites (1). As the mammalian embryo is difficult to access within the uterus for experimental manipulation, mouse mutants are invaluable tool for investigation of developmental mechanisms including those that take place in the caudal part of the mouse embryo. Development of the caudal part of the truncate and splotch mouse mutant, where tail development is affected, was analyzed. Splotch was first described in 1947, as a spontaneous mouse mutation of Pax3 gene affecting embryo development (2). Splotch homozygotes die during embryo development showing disorders of neural tube (spina bifida, exencephaly), abnormalities of neural crest cells (absence of sympathetic and spinal ganglia, Schwann cells, melanocytes and cells which migrate to the heart) and defects in somite structure. Corresponding mutation in humans is Waardenburg syndrome: defects of head development (deafness), hand musculature and localized absence of pigmentation. Truncate is a mouse strain with disorders in embryo development caused by spontaneously arisen mutation. Heterozygotes exhibit normal phenotype, while homozygotes have truncated tail, due to the partially absent notochord (3) Splotch and truncate homozygous embryos aged 11.5 and 12.5 days were isolated. They were fixed in a mixture of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide. One group of embryos was embedded in Durcopan (Fluka) and serial semithin sections, perpendicular to the longitudinal tail axis, were obtained, stained with toluidine blue and examined by light microscopy. Second group of embryos after fixation and postfixation were dehydrated in ascending mixtures of ethanol and amyl acetate to 100% amyl acetate, dried with liquid CO2, sputter coated with gold and observed in the scanning electron microscope. Splotch 11.5 days old embryos showed open neural tube in the most cranial third of the embryo tail. However, development of other axial structures was not influenced by abnormal neural tube, as all axial structures were normal and continuous with the tail bud. In contrast, splotch 12.5 days old embryos showed open neural tube accompanied with disturbed development of other axial structures and disturbed morphology of the tail bud. It is suggested that a longer presence of open neural tube influenced development of all structures caudal from the defect. Truncate homozygous embryos exhibited partially disturbed development of the notochord. In both 11.5 and 12.5 days old embryos, notochord was not continuous with the tail bud, but with the ventral neuroepithelium. Also, discontinuous or, even doubled notochord was found. Lack of the notochord in the caudal part of the tail was accompanied with disturbed development of the neural tube and the somites. Neural tube was smaller in diameter, it lacked lumen and massive cell dead in neuroepithelium was found. In parts of the tail without notochord, somites were connected in the midline of the tail. The observed malformations indicate that the normal sequence of tail development requires the coordinated interactions among the tail axial structures, which can be related to pathogenesis of spina bifida in humans.

The morphogenesis of the caudal part of the embryo includes formation of a mass of undifferentiated cells in the posterior end of the tail, referred as the tail bud. In the cranial direction the tail bud is continuous to the neural tube, notochord and the tail gut, while paraaxial mesenchyme segments in the somites (1). Development of the caudal part of the neural tube in truncate and splotch mouse mutants, where tail development is affected, was analyzed. As the mammalian embryo is difficult to access within the uterus for experimental manipulation, mouse mutants are invaluable tool for investigation of developmental mechanisms including those that take place in the caudal part of the mouse embryo. Splotch was first described in 1947, as a spontaneous mouse mutation of Pax3 gene affecting embryo development (2). Splotch homozygotes die during embryo development showing disorders of neural tube (spina bifida, exencephaly), abnormalities of neural crest cells (absence of sympathetic and spinal ganglia, Schwann cells, melanocytes, and cells which migrate to the heart) and defects in somite structure. Corresponding mutation in humans is Waardenburg syndrome: defects of head development (deafness), hand musculature and localized absence of pigmentation. Truncate is a mouse strain with disorders in embryo development caused by spontaneously arisen mutation. Heterozygotes exhibit normal phenotype, while homozygotes have truncated tail, due to the partially absent notochord (3). Splotch and truncate homozygous embryos aged 11.5 and 12.5 days were isolated. They were fixed in a mixture of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide. One group of embryos was embedded in Durcopan (Fluka) and serial semithin sections, perpendicular to the longitudinal tail axis, were obtained, stained with toluidine blue and examined by light microscopy. Second group of embryos after fixation and postfixation were dehydrated in ascending mixtures of ethanol and amyl acetate to 100% amyl acetate, dried with liquid CO2, sputter coated with gold and observed in the scanning electron microscope. Splotch 11.5 days old embryos showed open neural tube in the most cranial third of the embryo tail. However, development of other axial structures was not influenced by abnormal neural tube, as all axial structures were normal and continuous with the tail bud. In contrast, splotch 12.5 days old embryos showed open neural tube accompanied with disturbed morphology of the tail bud and irregularities of the caudal ends of the axial structures. Truncate homozygous embryos exhibited partially disturbed development of the notochord. In both 11.5 and 12.5 days old embryos, notochord was fragmented. In the parts of the tail, where notochord was missing, neural tube showed a variable morphology. Near the base of the tail neural tube morphology was comparable to the controls while in the distal regions it lacked floor plate and developing spinal ganglia were displaced in ventral direction. Occasionally, in the tip of the tail extensive cell death was present and secondary neurulation was disturbed. The observed malformations indicated that the normal sequence of tail development required coordinated interactions among tail axial structures. In splotch primary defect of the neural tube affected development of the tail bud and the neighboring structures, while opposite to that in truncate embryos discontinuities of the notochord changed the morphology of the neural tube.

Gene trap mutagenesis as a tool for identification and functional analysis of mouse genes expressed in the central nervous system

Nucleolar protein 1 was found as a marker of nucleolus in highly proliferated cells. It is present in majority of malignant tissues (lung, breast, prostate, colon, and brain), but also in non-malignant, highly proliferative tissues (PHA treated lymphocytes). High expression of Nol1 in different carcinoma is a marker of poor clinical prognosis. Nol1 expression is regulated during cell cycle: its protein synthesis starts at late G1, and peaks in S phase of cell cycle. In order to investigate expression and function of Nol1 gene, the mouse carrying a gene trap modification of Nol1 gene was produced. Embryonic stem cell genome was modified by insertion of nonhomologous DNA construct carrying selector (neoR) and marker (lacZ). From selected embryonic stem cells, animal carrying the corresponding gene modification was generated. Modified gene is tagged and its expression can be monitored by lacZ activity. Due to inserted construct sequence, function of trapped gene could be partially or totally lost, hence function of the gene can be elucidated. The expression of Nol1 gene during development was determined by whole mount histochemical staining by beta-galactosidase substrate X-gal. Staining was found all over the embryo and in all investigated developmental stages. The strongest staining was found between stages E8.5 and E12.5. Although all tissues expressed Nol1, it was not present in all cells. At the cellular level, majority of staining was found within the nucleolus. In order to get insight in the function of Nol1, intercross of heterozygous mice carrying gene trap insertion was performed. Using the PCR method we demonstrated that homozygous mice were not present among newborn animals. Therefore we assumed that homozygous phenotype is lethal.

The caudal part of the vertebrate embryo develops from the undifferentiated blastema located in the tip of the embryonic tail referred as the tail bud. The formation of the tail structures: the neural tube, the notochord and the tail gut does not involve folding of epithelial sheets and cell migration characteristic for gastrulation. The tail structures arise by rearrangement of mesenchymal cells of the tail bud without formation of the germ layers, i.e. by secondary body formation. In order to investigate mechanisms of tail formation and the role of T and Pax3 genes in this process, we analyzed three mouse mutants with tail malformations: brachyury, splotch and truncate. The normal and mutant embryos were analyzed by serial semithin sections, scanning and transmission electron microscopy. All three mutants showed aberrant tail bud differentiation. Brachyury heterozygous embryos develop the duplication of the tail tip containing two tail buds. Splotch homozygotes show proper formation of the notochord and the tail gut, but the caudal part of the secondary neural tube lacks the lumen and it is represented by in irregular group of cells. In the lumbosacral region the neural tube is opened. In the truncate homozygous embryos the tail notochord is partially missing. In contrast to the normal embryos, where the notochord develops together with the tail gut, in truncate it detaches from the wall of the neural tube. The observed malformations indicate that the proper differentiation of the tail bud is necessary for the development of the caudal part of the embryo.